This invention is related to the field of analytical techniques. In particular it is related to the area of sample analysis to determine the presence of pathogens.
Infections caused by staphylococci remain an important cause of morbidity and mortality. The virulence factors associated with the toxinogenic diseases of Staphylococcus aureus are the staphylococcal enterotoxins. Among them staphylococcal enterotoxin B (SEB) attracts the most attention because of its implication in immunological reactions. SEB has been shown to be able to stimulate mitogenic activity in T-cells (1). This phenomenon appears to involve specific binding of the toxin to major histocompatibility complex (MHC) class II molecules and subsequent stimulation of the T-cell via the TCR-V-beta elements (2).
Glycosphingolipids (GSL) are composed of carbohydrates, fatty acid and sphingosine. They are components of the eukaryotic cell membrane. Recently, GSL have been implicated in various biological phenomena. For example, GSL have been shown to be involved in cell proliferation (3, 4), cell migration (5, 6), and apoptosis (programmed cell death) (7). Most importantly, GSL have been shown to serve as receptors for numerous bacterial toxins and viruses (8-10). For example, a ganglioside GM1, has been long established to serve as a receptor for cholera toxin (11).
We have recently shown that digalactosylceramide (diGalCer) present in the human kidney and proximal tubular cells can specifically bind SEB. It did not bind structurally related toxins, staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin-1 (TSST-1) (12). The specificity of binding to the diGalCer receptor and physiological function was established subsequently. We found that SEB induced the uptake of [14C]choline and increased the synthesis of phosphatidylcholine, in contrast SEA and TSST-1 failed to stimulate phospholipid biosynthesis (13).
There is a need in the art for improved assays for detecting pathogens which can be used outside of laboratories in the absence of sophisticated equipment.
It is an object of the present invention to provide a method for assaying a test sample for the presence of a pathogen.
It is another object of the present invention to provide a method for assaying a test sample for the presence of a toxin.
It is an object of the present invention to provide a method for assaying a test sample for the presence of Staphylococcal enterotoxin-B.
It is another object of the invention to provide a kit for detecting Staphylococcal enterotoxin-B.
It is still another object of the invention to provide a kit for detecting a bacterial pathogen in a sample.
It is another object of the invention to provide a method for assaying for lymphocytes, neutrophils, or platelets.
It is another object of the invention to provide a method for assaying for cancer cells, including colon carcinoma and Burkitt""s tumor.
These and other objects of the invention are achieved by providing one or more of the embodiments described below. In one embodiment a method for ansaying a test sample for the presence of a pathogen is provided. The method comprises the steps of:
applying a glycosphingolipid to a polyvinylidene difluoride (PVDF) surface; wherein the glycosphingolipid is a receptor for a characteristic component of the pathogen; wherein the glycosphingolipid specifically binds the characteristic component;
applying a liquid reaction medium comprising the test sample to the PVDF surface;
removing excess liquid medium from the PVDF surface;
incubating the PVDF surface with an antibody which specifically binds to the characteristic component;
detecting the presence of the antibody on the PVDF surface, wherein the presence of the antibody indicates the presence of the characteristic component in the test sample.
According to another aspect of the invention a method is provided for assaying a test sample for the presence of a toxin. The method comprises the steps of:
applying a protein to a PVDF surface; wherein the protein is a receptor for the toxin, wherein the protein specifically binds the toxin;
applying a liquid reaction medium comprising the test sample to the PVDF surface;
removing excess liquid medium from the PVDF surface;
incubating the PVDF surface with an antibody which specifically binds to the toxin;
detecting the presence of the antibody on the PVDF surface, wherein the presence of the antibody indicates the presence of the toxin in the test sample.
According to still another aspect of the invention, a method is provided for assaying a test sample for the presence of a Staphylococcal enterotoxin-B. The method comprises the steps of:
applying digalactosylceramide to a PVDF surface;
applying a liquid reaction medium comprising the test sample to the PVDF surface;
removing excess liquid medium from the PVDF surface;
incubating the PVDF surface with an antibody which specifically binds to Staphylococcal enterotoxin-B;
detecting the presence of the antibody on the PVDF surface, wherein the presence of the antibody indicates the presence of Staphylococcal enterotoxin-B in the test sample.
In yet another embodiment of the invention a kit is provided for the detection of a pathogen. The kit comprises:
a PVDF surface;
a purified sample of a glycosphingolipid which specifically binds to a characteristic component of the pathogen;
an antibody which specifically binds to the characteristic component of the pathogen.
In yet another embodiment of the invention a kit is provided for the detection of Staphylococcal enterotoxin-B. The kit comprises:
a PVDF surface;
a purified sample of digalactosylceramide;
an antibody which specifically binds to Staphylococcal enterotoxin-B.
According to yet another aspect of the invention a method is provided for assaying a test sample for lymphocytes, neutrophils, or platelets. The method comprises the steps of:
applying a glycoprotein to a PVDF surface; wherein the glycoprotein is selected from the group consisting of L-selectin, E-selectin, and P-selectin;
applying a liquid test medium which may comprise neutrophils, lymphocytes, or platelets to the PVDF surface;
removing excess liquid medium from the PVDF surface after incubation;
incubating the PVDF surface with an antibody which specifically binds to the glycoprotein;
detecting the presence of the antibody on the PVDF surface, wherein the presence of the antibody indicates the presence of neutrophils, lymphocytes, or platelets in the test sample.
In another aspect of the invention a method for assaying a test sample for the presence of colon carcinoma is provided. The method comprises the steps of:
applying a test sample to a PVDF surface, wherein the test sample comprises a biopsy of suspected colon carcinoma, a lipid extract of said biopsy, or a urine sample of a patient suspected of having colon carcinoma;
washing the PVDF surface to remove components which do not bind;
incubating the PVDF surface with an antibody which specifically binds to digalactosylceramide;
detecting the presence of the antibody on the PVDF surface, wherein the presence of the antibody indicates the presence of colon carcinoma in the patient who provided the test sample.
According to another aspect of the invention a method for assaying a test sample for the presence of Burkitt""s tumor is provided. The method comprises the steps of:
applying a test sample to a PVDF surface, wherein the test sample comprises a biopsy of suspected Burkitt""s tumor, a lipid extract of said biopsy, or a urine sample of a patient suspected of having Burkitt""s tumor;
washing the PVDF surface to remove components which do not bind;
incubating the PVDF surface with an antibody which specifically binds to globotriosylceramide;
detecting the presence of the antibody on the PVDF surface, wherein the presence of the antibody indicates the presence of Burkitt""s tumor in the patient who provided the test sample.
The present invention thus provides the art with an assay which is widely applicable because it is highly specific, it does not require special equipment, and the results can be obtained within few hours with the naked eye. Moreover, since the glycosphingolipid receptors have a long-shelf life, they can be easily stored and used for a long time. In addition, the use of non-radioactive reagents renders this assay safer and more widely applicable.